The turn of the twentieth century has witnessed the continued appearance of new pathogens as well as the development of antibiotic resistance and re-emergence of old pathogens. Despite the enormous burden that bacterial pathogens impose on human health, there is relatively little understanding of host-pathogen interactions. The hypothesis underpinning this grant proposal is that by studying the mechanisms by which bacteria enter Drosophila cells, replicate and ultimately kill the cells, we will be able to identify evolutionary conserved host factors that are involved in the interaction between pathogens and their hosts. Studies have clearly established that Drosophila S2 cells, which have the properties of macrophages, are an excellent system to study host-pathogen interactions. We will focus our studies on Mycobacterium spp. It is estimated that one-third of the world's population is infected with Mycobacterium tuberculosis, and it kills more people yearly than any other bacterial pathogen. It, like other pathogenic mycobacteria, can convert the normally hostile environment of a macrophage into a niche in which it can effectively replicate. We propose high-throughput full genome screens in Drosophila cells, using the RNA interference (RNAi) methodology, to identify host factors required for entry into and survival within macrophages. Preliminarydata indicatethat M.fortuitum and M. marinum effectively enter and replicate in Drosophila cells, and that we can identify host factors required for these processes. We hypothesize that by elucidating the host factors required for mycobacterial entry and survival within host cells, we will gain an understandingof the common machinery used by intracellular pathogens as well as specific factors that are important for the intracellular strategy employed by mycobacteria. The host factors identified in Drosophila cells will then be used to dissect the mechanisms of bacterial uptake and intracellular survival followed by validation in mammalian systems. Thus, exploiting the powerful genetic strategy of RNAi in Drosophila, we hope to elucidate novel bacterial strategies for intracellular survival that may ultimately allow us to gain functional and mechanistic insight into the interaction between M. tuberculosis and its human host.